Molecular array generation using photoresist

This application claims priority to U.S. Provisional Patent Application No. 63/132,385, filed Dec. 30, 2020, entitled “MOLECULAR ARRAY GENERATION USING PHOTORESIST,” which is herein incorporated by reference in its entirety for all purposes.

The present disclosure relates in some aspects to methods for manufacturing a molecular array using photoresist and the molecular array generated in situ on a substrate.

Arrays of nucleic acids are an important tool in the biotechnology industry and related fields. These nucleic acid arrays, in which a plurality of distinct or different nucleic acids are positioned on a solid support surface in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like.

A feature of many arrays that have been developed is that each of the distinct nucleic acids of the array is stably attached to a discrete location on the array surface, such that its position remains constant and known throughout the use of the array. Stable attachment is achieved in a number of different ways, including covalent bonding of a nucleic acid polymer to the support surface and non-covalent interaction of the nucleic acid polymer with the support surface.

There are two main ways of producing nucleic acid arrays in which the immobilized nucleic acids are covalently attached to the substrate surface, i.e., via in situ synthesis in which the nucleic acid polymer is grown on the surface of the substrate in a step-wise, nucleotide-by-nucleotide fashion, or via deposition of a full, presynthesized nucleic acid/polypeptide, cDNA fragment, etc., onto the surface of the array.

While nucleic acid arrays have been manufactured using in situ synthesis techniques, applications in the field of genomics and high throughput screening have fueled the demand for precise chemistry and high fidelity of the synthesized oligonucleotides. Accordingly, there is continued interest in the development of new methods for producing nucleic acid arrays in situ. Provided herein are methods, uses and articles of manufacture that meet such needs.

In some aspects, disclosed herein is a method for providing an array, comprising: (a) irradiating a substrate comprising an unmasked first region and a masked second region, whereby a photoresist in the first region is degraded to render oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas oligonucleotide molecules in the second region are protected by a photoresist in the second region from hybridization and/or ligation; and (b) attaching an oligonucleotide of at least four nucleotide residues in length to oligonucleotide molecules in the first region via hybridization and/or ligation, wherein oligonucleotide molecules in the second region do not receive a sequence of the oligonucleotide, thereby providing on the substrate an array comprising different oligonucleotide molecules in the first and second regions. In some embodiments, the oligonucleotide comprises a barcode sequence, and all or a portion of the barcode sequence is attached to oligonucleotide molecules in the first region, wherein oligonucleotide molecules in the second region do not receive the barcode sequence or portion thereof.

In any of the embodiments herein, the oligonucleotide molecules on the substrate can comprise one or more common sequences. In any of the embodiments herein, the one or more common sequences can comprise a homopolymeric sequence, such as a poly(dT) sequence, of three, four, five, six, seven, eight, nine, ten or more nucleotide residues in length. In any of the embodiments herein, the one or more common sequences can comprise a common primer sequence. In some embodiments, the common primer sequence is between about 10 and about 35 nucleotides in length. In any of the embodiments herein, the one or more common sequences can comprise a partial primer sequence. For example, a terminal sequence of an oligonucleotide molecule on the substrate together with a sequence of an oligonucleotide attached to the oligonucleotide molecule on the substrate can form the hybridization sequence for a primer. In this example, the terminal sequence of the oligonucleotide molecule on the substrate can be viewed as a partial primer sequence. In any of the embodiments herein, oligonucleotide molecules in the first region and oligonucleotide molecules in the second region can be identical in sequence. In any of the embodiments herein, oligonucleotide molecules on the substrate prior to the irradiating step can be identical in sequence. In any of the embodiments herein, oligonucleotide molecules in the first region and oligonucleotide molecules in the second region can be different in sequences, optionally wherein oligonucleotide molecules in the first region and oligonucleotide molecules in the second region comprise different barcode sequences. In any of the embodiments herein, oligonucleotide molecules on the substrate can comprise two or more different sequences, optionally wherein oligonucleotide molecules on the substrate can comprise two, three, four, five, six, seven, eight, nine, ten or more different barcode sequences.

In any of the embodiments herein, oligonucleotide molecules on the substrate can be immobilized in a plurality of features. In any of the embodiments herein, the 3′ terminal nucleotides of the immobilized oligonucleotide molecules can be distal to the substrate or array surface. In any of the embodiments herein, the 5′ terminal nucleotides of the immobilized oligonucleotide molecules can be more proximal to the substrate or array surface than the 3′ terminal nucleotides. In any of the embodiments herein, one or more nucleotides at or near the 5′ terminus of each immobilized oligonucleotide can be directly or indirectly attached to the substrate or array surface, thereby immobilizing the oligonucleotides. In any of the embodiments herein, the 3′ terminus of each immobilized oligonucleotide can project away from the substrate or array surface. In any of the embodiments herein, the 5′ terminal nucleotides of the immobilized oligonucleotide molecules can be distal to the substrate or array surface. In any of the embodiments herein, the 3′ terminal nucleotides of the immobilized oligonucleotide molecules can be more proximal to the substrate or array surface than the 5′ terminal nucleotides. In any of the embodiments herein, one or more nucleotides at or near the 3′ terminus of each immobilized oligonucleotide can be directly or indirectly attached to the substrate or array surface, thereby immobilizing the oligonucleotides. In any of the embodiments herein, the 5′ terminus of each immobilized oligonucleotide can project away from the substrate or array surface.

In any of the embodiments herein, oligonucleotide molecules on the substrate prior to the irradiating step can be between about 4 and about 100 nucleotides in length. In any of the embodiments herein, oligonucleotide molecules on the substrate prior to the irradiating step can be between about 10 and about 50 nucleotides in length.

In any of the embodiments herein, oligonucleotide molecules on the substrate can comprise functional groups, optionally wherein the functional groups are amino or hydroxyl groups. In any of the embodiments herein, prior to the irradiating step, the functional groups do not need to be protected by a protective group that is removed during and/or after the irradiating step. In any of the embodiments herein, prior to the irradiating step, the functional groups can be unprotected functional groups. In any of the embodiments herein, prior to the irradiating step, the functional groups can be amino or hydroxyl groups that are protected by the photoresist but are not reacted with protective groups. In any of the embodiments herein, prior to the irradiating step, the functional groups can be amino or hydroxyl groups that are not reacted with a protective group, moiety, or molecule that is photo-sensitive, photo-labile, photo-degradable, acid-labile or otherwise removable in a photo-activable reaction. In any of the embodiments herein, the functional groups can be 3′ hydroxyl groups of nucleotides.

In any of the embodiments herein, the method can further comprise a step of providing the substrate, wherein the first and second regions have the same photoresists. In any of the embodiments herein, the providing step can comprise applying the photoresist to the substrate, thereby forming a photoresist layer on the substrate. In some embodiments, the photoresist can be applied to the substrate via spin coating and/or dipping. In any of the embodiments herein, oligonucleotide molecules on the substrate can be embedded in the photoresist. In any of the embodiments herein, oligonucleotide molecules on the substrate can be embedded in an underlayer, and the photoresist can form a photoresist layer on top of the underlayer. In any of the embodiments herein, the underlayer can be a soluble polymer.

In any of the embodiments herein, the method can further comprise forming a pattern of oligonucleotide molecules on the substrate prior to applying the photoresist to the substrate. In any of the embodiments herein, the forming step can comprise: irradiating a substrate comprising a plurality of functional groups and a photoresist through a patterned mask, whereby the photoresist in a first region of the substrate is degraded, rendering functional groups in the first region available for reacting with functional groups in functionalized oligonucleotide molecules, whereas functional groups in a second region of the substrate are protected by the photoresist from reacting with functional groups in the functionalized oligonucleotide molecules; and contacting the substrate with the functionalized oligonucleotide molecules, wherein the functionalized oligonucleotide molecules are coupled to functional groups in the first region but not to functional groups in the second region, thereby forming a pattern of oligonucleotide molecules on the substrate.

In any of the embodiments herein, the plurality of functional groups of the substrate (e.g., of a lawn of oligonucleotides) can remain unreacted functional groups (e.g., unreacted amino or hydroxyl groups that are protected by a photoresist from nucleic acid hybridization and/or ligation) prior to, during, and/or after the irradiating step, until the attachment of the oligonucleotide of at least four nucleotide residues in length. In any of the embodiments herein, the plurality of functional groups do not need to be deprotected in order to interact and/or react with the oligonucleotide of at least four nucleotide residues in length. In any of the embodiments herein, prior to the irradiating step, the plurality of functional groups of the substrate (e.g., of a lawn of oligonucleotides) do not need to be protected by a protective group, moiety, or molecule that is photo-sensitive, photo-labile, photo-degradable, acid-labile or otherwise removable in a photo-activable reaction.

In any of the embodiments herein, the plurality of functional groups of the substrate (e.g., of a lawn of oligonucleotides) can be protected prior to the irradiating step and deprotected during and/or after the irradiating step. In any of the embodiments herein, prior to the irradiating step, the plurality of functional groups of the substrate (e.g., of a lawn of oligonucleotides) can be protected by a protective group, moiety, or molecule that is photo-sensitive, photo-labile, photo-degradable, acid-labile or otherwise removable in a photo-activable reaction. In any of the embodiments herein, the protective group, moiety, or molecule can be removed, thereby deprotecting the plurality of functional groups and allowing them to interact and/or react with the oligonucleotide of at least four nucleotide residues in length.

In any of the embodiments herein, the plurality of functional groups (e.g., of a lawn of oligonucleotides) can be or comprise amino groups, hydroxyl groups, aldehyde groups, and/or click chemistry groups, optionally wherein the click chemistry groups are capable of a nucleophilic addition reaction, a cyclopropane-tetrazine reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, an alkyne hydrothiolation reaction, an alkene hydrothiolation reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron-demand Diels-Alder (IED-DA) reaction, a cyanobenzothiazole condensation reaction, an aldehyde/ketone condensation reaction, or a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

In any of the embodiments herein, the functional groups in the functionalized oligonucleotide molecules can be or comprise amino groups. In any of the embodiments herein, the functionalized oligonucleotide molecules can be 5′ amine-terminated.

In any of the embodiments herein, the method can further comprise heating the substrate to dryness during or after the contacting step. In any of the embodiments herein, the method can further comprise blocking unreacted functional groups of the substrate.

In any of the embodiments herein, the method can further comprise rendering the reaction between functional groups of the substrate and the functionalized oligonucleotide molecules irreversible.

In any of the embodiments herein, aldehyde groups of the substrate can be reacted with 5′ amino groups of the functionalized oligonucleotide molecules, and the substrate can be contacted with a reagent to block unreacted aldehyde groups and render the reaction irreversible. In any of the embodiments herein, the reagent can be or comprise sodium borohydride. In any of the embodiments herein, the reagent (e.g., sodium borohydride) can block unreacted aldehyde groups via reductive amination of an aldehyde-amine condensation reaction product.

In any of the embodiments herein, the irradiating and contacting steps can be repeated in one or more cycles, e.g., each cycle for coupling functionalized oligonucleotide molecules to functional groups in one or more regions of the substrate. In some embodiments, each cycle is for coupling functionalized oligonucleotide molecules to functional groups in a different region of the substrate. In some embodiments, the different regions of the substrate do not overlap. In any of the embodiments herein, the photoresist does not need to be removed prior to, during, or between the one or more cycles, optionally wherein the method does not comprise re-applying a photoresist to the substrate prior to, during, or between the one or more cycles. In any of the embodiments herein, the photoresist can be removed in a cycle and re-applied in the next cycle, and the removed photoresist and the re-applied photoresist can be the same or different. In any of the embodiments herein, the photoresist does not need to be removed prior to, during, or after each cycle or between cycles. In some embodiments, the photoresist remains on the substrate for a plurality of cycles and is removed after the plurality of cycles and re-applied prior to the next cycle. In some embodiments, the photoresist re-applied to the substrate is the same photoresist. In another embodiment, the photoresist re-applied to the substrate is a different photoresist.

In any of the embodiments herein, the photoresist in the first and/or second regions can comprise a photoacid generator. In any of the embodiments herein, the photoresist can comprise an acid scavenger. In any of the embodiments herein, the photoresist can comprise a base quencher. In any of the embodiments herein, the photoresist can comprise a photosensitizer. In any of the embodiments herein, the photoresist can comprise a surfactant and/or a casting solvent.

In any of the embodiments herein, the substrate can be irradiated with a UV light. In any of the embodiments herein, the substrate can be irradiated through a patterned mask. In any of the embodiments herein, the method can further comprise removing the patterned mask after the irradiating step, optionally wherein the same patterned mask can be re-used in a subsequent cycle of the irradiating and contacting steps, wherein the patterned mask is moved (e.g., rotated, for example, by 90 degrees) relative to the substrate; or optionally wherein a different patterned mask is used in a subsequent cycle of the irradiating and contacting steps. In any of the embodiments herein, the photoresist of the substrate can be dissolved by a developer and removed. In any of the embodiments herein, the photoresist in the first region of the substrate can be dissolved by a first developer and removed. In any of the embodiments herein, the photoresist in the second region of the substrate can be dissolved by a second developer and removed.

In any of the embodiments herein, the barcode sequence or any part thereof can be between about 4 and about 50 nucleotides in length. In any of the embodiments herein, the barcode sequence or any part thereof can be between about 5 and about 25 nucleotides in length.

In any of the embodiments herein, the oligonucleotide comprising the barcode sequence can be between about 10 and about 50 nucleotides in length. In any of the embodiments herein, the oligonucleotide comprising the barcode sequence can be hybridized to an oligonucleotide molecule in the first region. In any of the embodiments herein, the oligonucleotide comprising the barcode sequence can be ligated to an oligonucleotide molecule in the first region.

In any of the embodiments herein, the oligonucleotide comprising the barcode sequence can be hybridized to a splint which is in turn hybridized to an oligonucleotide molecule in the first region. In any of the embodiments herein, the method can further comprise ligating the oligonucleotide comprising the barcode sequence to the oligonucleotide molecule to generate a barcoded oligonucleotide molecule in the first region. In any of the embodiments herein, the oligonucleotide and the oligonucleotide molecule can be ligated using the splint as template, without or without gap filling prior to the ligation.

In any of the embodiments herein, the method can further comprise removing the splint after the ligation. In any of the embodiments herein, the splint can be removed by heat and/or treatment with a denaturing agent, such as KOH or NaOH. In any of the embodiments herein, the method can further comprise blocking the 3′ or 5′ termini of barcoded oligonucleotide molecules and/or unligated oligonucleotide molecules in the first region from ligation. In any of the embodiments herein, the blocking can comprise adding a 3′ dideoxy, a non-ligating 3′ phosphoramidate, or a triphenylmethyl (trityl) group to the barcoded oligonucleotide molecules and/or unligated oligonucleotide molecules, optionally wherein the blocking by the trityl group is removed with a mild acid after ligation is completed. In any of the embodiments herein, the addition can be catalyzed by a terminal transferase, e.g., TdT. In any of the embodiments herein, the blocking can be removed using an internal digestion of the barcoded oligonucleotide molecules after ligation is completed.

In any of the embodiments herein, the method can comprise N cycles, wherein Nis an integer of 2 or greater, and one or more or all of the N cycles comprises the irradiating and the attaching steps. In any of the embodiments herein, the irradiating and the attaching steps can be repeated N cycles, each cycle for one or more regions of the substrate (e.g., for one or more features on an array), for a round until all desired regions have been exposed to light, deprotected from the photoresist once, and oligonucleotide molecules in the exposed regions have received a barcode sequence for that round, which barcode sequence may be the same or different for molecules for any two given regions (e.g., features on an array). The barcode sequences for different cycles (e.g., each cycle for a different region of the substrate) in the same round can comprise the same or different sequences, and preferably the barcode sequences for different cycles are different. In any of the embodiments herein, the barcode sequences received by oligonucleotide molecules in feature(s) on the substrate in cycle I and in feature(s) in cycle J can be different, wherein I and J are integers and 1≤I<J≤N. In any of the embodiments herein, the photoresist may not need to be removed prior to, during, or between one or more of the N cycles. In any of the embodiments herein, a photoresist may not need to be re-applied to the substrate prior to, during, or between one or more of the N cycles of a given round. In any of the embodiments herein, the method can further comprise removing photoresist from the substrate after each cycle and re-applying photoresist to the substrate prior to the subsequent cycle of the same round or the first cycle of a subsequent round.

In any of the embodiments herein, the method can comprise M rounds, wherein M is an integer of 2 or greater, and each of the M rounds comprises one or more cycles. In any of the embodiments herein, each of the M rounds may comprise N cycles, optionally wherein each cycle is for attaching oligonucleotides to oligonucleotide molecules in one or more regions of the substrate (e.g., for one or more features on the array). In any of the embodiments herein, the method can further comprise removing photoresist from the substrate after each round or after one or more or all of a plurality of rounds (e.g., after two, three, or more sequential rounds) and re-applying photoresist to the substrate prior to a new round. In any of the embodiments herein, each of the M rounds can comprise N cycles, wherein N is 3 or greater. In any of the embodiments herein, each of the M rounds can comprise the same number of cycles, or two or more of the M rounds can comprise different numbers of cycles. With reference to, although the figure shows Cycle 1, Cycle 2, . . . , Cycle N for each of Round 1 and Round M, it should be appreciated that any two rounds of Round 1 to Round M may comprise the same number or different numbers of sequential cycles. For instance, Round 2 may comprise fewer than N cycles, whereas Round 3 may comprise more than N cycles. For instance, Cycle 1 and Cycle 2 of Round 2 may be combined into one cycle and the regions in these cycles receive the same oligonucleotide, and in Round 3 the regions after Cycle (N−1) may be grouped into two sets, one set for Cycle N and the other set for Cycle (N+1), and each set may receive a different oligonucleotide. One or more rounds comprising the attachment of a common nucleic acid sequence may be performed before or after any of Round 1 to Round M, and the nucleic acid sequence can be common to two or more regions on the substrate. In some cases, the nucleic acid sequence can be universal and can be shared by all of the regions on the substrate.

In any of the embodiments herein, oligonucleotide molecules in a feature of the substrate can receive a first barcode sequence in one of the cycles in round K, wherein K is an integer and 1≤K<M, and oligonucleotide molecules in the feature comprising the first barcode sequence receive a second barcode sequence in one of the cycles in round (K+1), thereby forming oligonucleotide molecules comprising the first and second barcode sequences. In any of the embodiments herein, the diversity of barcode sequences in the oligonucleotides in a plurality of features on the substrate can be N. In any of the embodiments herein, the feature(s) can be no more than 0.5 micron, no more than 1 micron, no more than 5 microns, no more than 7 microns, no more than 10 microns, or no more than 15 microns, no more than 20 microns, no more than 25 microns, no more than 30 microns, or no more than 35 microns, no more than 40 microns, no more than 45 microns, or no more than 50 microns in diameter. In any of the embodiments herein, the feature(s) can be no more than 500 nm, no more than 600 nm, no more than 700 nm, no more than 800 nm, no more than 900 nm, no more than 1 micron, no more than 1.5 microns, no more than 2 microns, no more than 2.5 microns, no more than 3 microns, no more than 3.5 microns, no more than 4 microns, no more than 4.5 microns, or no more than 5 microns in one dimension. In any of the embodiments herein, the feature(s) can be no more than 500 nm, no more than 600 nm, no more than 700 nm, no more than 800 nm, no more than 900 nm, no more than 1 micron, no more than 1.5 microns, no more than 2 microns, no more than 2.5 microns, no more than 3 microns, no more than 3.5 microns, no more than 4 microns, no more than 4.5 microns, or no more than 5 microns in two dimensions.

In some aspects, provided herein is a method for providing an array, comprising: (a) irradiating a substrate comprising an unmasked first region and a masked second region, whereby a photoresist in the first region is degraded to render oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas oligonucleotide molecules in the second region are protected by the photoresist in the second region from hybridization and/or ligation; and (b) contacting oligonucleotide molecules in the first region with a first splint and a first oligonucleotide comprising a first barcode sequence, wherein the first splint hybridizes to the first oligonucleotide and one of the oligonucleotide molecules in the first region, wherein the first oligonucleotide is ligated to the oligonucleotide molecules in the first region, and the first oligonucleotide is not ligated to oligonucleotide molecules in the second region, thereby providing on the substrate an array comprising different oligonucleotide molecules in the first and second regions.

In any of the embodiments herein, the hybridization region between the first splint and one of the oligonucleotide molecules can be at least 3, 4, 5, 6, 7, 8, 9, 10 bp or more than 10 bp. In any of the embodiments herein, the hybridization region between the first splint and the first oligonucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10 bp or more than 10 bp.

In any of the embodiments herein, hybridization to the first splint can bring the terminal nucleotides of the first oligonucleotide and one of the oligonucleotide molecules immediately next to each other, and the ligation of the juxtaposed first oligonucleotide and oligonucleotide molecules on the substrate does not require gap-filling. In any of the embodiments herein, hybridization to the first splint can bring the terminal nucleotides of the first oligonucleotide and one of the oligonucleotide molecules in proximity to each other and separated by one or more nucleotides, and the ligation of the juxtaposed first oligonucleotide and oligonucleotide molecules on the substrate is preceded by gap-filling.

In any of the embodiments herein, the photoresist can be a first photoresist, and the first oligonucleotide is ligated to the oligonucleotide molecules in the first region to generate first extended oligonucleotide molecules, and the method can further comprise: (c) applying a second photoresist to the substrate, optionally wherein the second photoresist is applied after the first photoresist is removed from the substrate; (d) irradiating the substrate while the first region is masked and the second region is unmasked, whereby the first and/or second photoresist in the second region is degraded to render oligonucleotide molecules in the second region available for hybridization and/or ligation, whereas the first extended oligonucleotide molecules in the first region are protected by the second photoresist in the first region from hybridization and/or ligation; and (e) contacting oligonucleotide molecules in the second region with a second splint and a second oligonucleotide comprising a second barcode sequence, wherein the second splint hybridizes to the second oligonucleotide and one of the oligonucleotide molecules in the second region, wherein the second oligonucleotide is ligated to the oligonucleotide molecules in the second region to generate second extended oligonucleotide molecules, and the second oligonucleotide is not ligated to the first extended oligonucleotide molecules in the first region.

In any of the embodiments herein, steps (a)-(b) can be part of a first cycle, steps (d)-(e) can be part of a second cycle, and steps (a)-(e) can be part of a first round (comprising the first and second cycles), and wherein the method can comprise one or more additional rounds, each with one or more cycles of the irradiating and the contacting steps. In any of the embodiments herein, steps (a)-(e) can be part of a first round, the first and second oligonucleotides can be Round 1 oligonucleotides, the first and second barcode sequences can be Round 1 barcode sequences, and the method can further comprise: (a′) irradiating the substrate while the first region is unmasked and the second region is masked, whereby a photoresist in the first region is degraded to render the first extended oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas the second extended oligonucleotide molecules in the second region are protected by the photoresist in the second region from hybridization and/or ligation; and (b′) attaching a first Round 2 oligonucleotide comprising a first Round 2 barcode sequence to the first extended oligonucleotide molecules in the first region via hybridization and/or ligation, wherein the second extended oligonucleotide molecules in the second region do not receive the first Round 2 barcode sequence.

In any of the embodiments herein, the photoresist can be a first photoresist, and the first Round 2 oligonucleotide can be ligated to the first extended oligonucleotide molecules in the first region to generate first further extended oligonucleotide molecules, and the method can further comprise: (c′) applying a second photoresist to the substrate, optionally wherein the second photoresist is applied after the first photoresist is removed from the substrate; (d′) irradiating the substrate while the first region is masked and the second region is unmasked, whereby the first or second photoresist in the second region is degraded to render the second extended oligonucleotide molecules in the second region available for hybridization and/or ligation, whereas the first further extended oligonucleotide molecules in the first region are protected by the second photoresist in the first region from hybridization and/or ligation; and (e′) attaching a second Round 2 oligonucleotide comprising a second Round 2 barcode sequence to the second extended oligonucleotide molecules in the second region via hybridization and/or ligation, wherein the first further extended oligonucleotide molecules in the first region do not receive the second Round 2 barcode sequence.

In other embodiments, the method can further comprise: (a′) irradiating the substrate while the second region is unmasked and the first region is masked, whereby a photoresist in the second region is degraded to render the second extended oligonucleotide molecules in the second region available for hybridization and/or ligation, whereas the first extended oligonucleotide molecules in the first region are protected by the photoresist in the first region from hybridization and/or ligation; and (b′) attaching a first Round 2 oligonucleotide comprising a first Round 2 barcode sequence to the second extended oligonucleotide molecules in the second region via hybridization and/or ligation, wherein the first extended oligonucleotide molecules in the first region do not receive the first Round 2 barcode sequence.

In some aspects, provided herein is a method for providing an array, comprising: (a) irradiating a substrate comprising an unmasked first region and a masked second region, whereby photoresist in the first region is degraded to render oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas oligonucleotide molecules in the second region are protected by photoresist in the second region from hybridization and/or ligation; and (b) attaching a first oligonucleotide of at least four residues in length (e.g., comprising a first barcode sequence) to oligonucleotide molecules in the first region via hybridization and/or ligation, wherein oligonucleotide molecules in the second region are not ligated to the first oligonucleotide or a portion thereof, thereby providing on the substrate an array comprising different oligonucleotide molecules in the first and second regions. In some aspects, the method further comprises (a′) irradiating the unmasked second region, whereby photoresist in the second region is degraded to render oligonucleotide molecules in the second region available for hybridization and/or ligation; (b′) attaching a second oligonucleotide of at least four residues in length (e.g., comprising a second barcode sequence) to oligonucleotide molecules in the second region via hybridization and/or ligation, whereas oligonucleotide molecules in the first region are not hybridized and/or ligated to the second oligonucleotide. For instance, oligonucleotide molecules in the first region may be protected by (i) photo-cleavable polymers bound to oligonucleotide molecules in the first region and/or (ii) photo-cleavable moieties of oligonucleotide molecules in the first region from hybridization and/or ligation, and/or splints can be used to hybridize to the second oligonucleotide and template ligation of the second oligonucleotide specifically to oligonucleotide molecules in the second region but not to oligonucleotide molecules in the first region based on sequence complementarity.

In some embodiments, the photoresist can be a first photoresist, and the first Round 2 oligonucleotide can be ligated to the second extended oligonucleotide molecules in the second region to generate second further extended oligonucleotide molecules, and the method can further comprise: (c′) applying a second photoresist to the substrate, optionally wherein the second photoresist is applied after the first photoresist is removed from the substrate; (d′) irradiating the substrate while the second region is masked and the first region is unmasked, whereby the first or second photoresist in the first region is degraded to render the first extended oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas the second further extended oligonucleotide molecules in the second region are protected by the second photoresist in the second region from hybridization and/or ligation; and (e′) attaching a second Round 2 oligonucleotide comprising a second Round 2 barcode sequence to the first extended oligonucleotide molecules in the first region via hybridization and/or ligation, wherein the second further extended oligonucleotide molecules in the second region do not receive the second Round 2 barcode sequence.

In any of the embodiments herein, the Round 1 barcode sequences can be different from each other. In any of the embodiments herein, the Round 2 barcode sequences can be different from each other. In any of the embodiments herein, the Round 1 barcode sequences can be different from the Round 2 barcode sequences.

In any of the embodiments herein, the oligonucleotide molecules on the substrate can comprise functional groups, optionally wherein the functional groups can be or comprise amino groups, hydroxyl groups, aldehyde groups, and/or click functional groups. In any of the embodiments herein, the functional groups do not need to be reacted with and/or protected by a chemical group, e.g., by a photo-sensitive protective group, moiety, or molecule. In any of the embodiments herein, the functional groups can be 3′ hydroxyl groups of nucleotides.

In some aspects, provided herein is a composition comprising: (i) a substrate comprising a first region and a second region, (ii) hybridization complexes in the first region, wherein at least one of the hybridization complexes comprise an oligonucleotide molecule immobilized in the first region hybridized to a first splint, which is in turn hybridized to a first oligonucleotide comprising a first barcode sequence, and (iii) oligonucleotide molecules immobilized in the second region and protected by a photoresist from hybridization and/or ligation.

In some embodiments, provided herein is a composition, comprising: (i) a substrate comprising a first region and a second region, (ii) hybridization complexes in the first region, wherein at least one of the hybridization complexes comprise an oligonucleotide molecule immobilized in the first region hybridized to a first splint, which is in turn hybridized to a first oligonucleotide comprising a first barcode sequence, wherein the hybridization complexes are protected by a first photoresist from hybridization and/or ligation, and (iii) oligonucleotide molecules immobilized in the second region and protected by a second photoresist from hybridization and/or ligation. In some embodiments, the first photoresist and the second photoresist are the same. In some embodiments, the first photoresist and the second photoresist are different. In any of the embodiments herein, the oligonucleotide molecules on the substrate can comprise functional groups, optionally wherein the functional groups can be amino or hydroxyl groups. In any of the embodiments herein, the functional groups may be unreacted or unprotected functional groups. For example, in some embodiments, the functional groups have not been reacted with and/or protected by a photo-sensitive group, moiety, or molecule. In some embodiments, the functional groups can be 3′ hydroxy groups of nucleotides.

In some embodiments, provided herein is a composition, comprising a substrate comprising a plurality of universal oligonucleotide molecules immobilized thereon, wherein the universal oligonucleotide molecules in a first region of the substrate are available for hybridization and/or ligation, and the universal oligonucleotide molecules in a second region of the substrate are embedded in a photoresist and protected from hybridization and/or ligation. In some embodiments, the composition further comprises a photomask masking the second region while exposing the first region to light. In some embodiments, the composition further comprises hybridization complexes in the first region, wherein at least one of the hybridization complexes comprise a universal oligonucleotide molecule immobilized in the first region hybridized to a first splint, which is in turn hybridized to a first oligonucleotide comprising a first barcode sequence. In some embodiments, the universal oligonucleotide molecules on the substrate can comprise functional groups. In some embodiments, the functional groups may be amino or hydroxyl groups. In some embodiments, the functional groups may not be protected. For example, in some embodiments, the functional groups may not be protected by a photo-sensitive group, moiety, or molecule. In some embodiments, the functional groups can be 3′ hydroxyl group of nucleotides.

In any of the embodiments herein, the composition can further comprise the photoresist. In the composition of any of the preceding embodiments, the photoresist can form a photoresist layer and oligonucleotide molecules immobilized in the second region can be embedded in the photoresist layer.

In any of the embodiments herein, the composition can further comprise a ligase capable of ligating the first oligonucleotide and the oligonucleotide molecule immobilized in the first region using the first splint as template, and optionally a polymerase capable of gap filling using the first splint as template prior to the ligation. In any of the embodiments herein, the composition may not comprise any dNTP or a polymerase capable of incorporating a dNTP into an oligonucleotide molecule. In any of the embodiments herein, the composition may not comprise any reagent for base-by-base oligonucleotide synthesis.

In any of the embodiments herein, a method disclosed herein may not comprises a step of contacting the substrate or oligonucleotide molecules immobilized thereon with any reagent for base-by-base oligonucleotide synthesis.

The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (comprising recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques comprise polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999),(Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007),; Dieffenbach, Dveksler, Eds. (2003),; Bowtell and Sambrook (2003), DNA; Mount (2004),; Sambrook and Russell (2006),; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995)(4th Ed.) W. H. Freeman, New York N.Y.; Gait, “” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,3rd Ed., W. H. Freeman Pub., New York, N.Y.; and Berg et al. (2002)5Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.